Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR6

The primary pathophysiological change giving rise to the symptoms of Parkinson's disease (PD) is a loss of the dopaminergic neurons in the substantia nigra pars compacta (SNc) that are involved in modulating the function of basal ganglia (BG) nuclei. Unfortunately, traditional therapies for treatment of PD based on dopamine replacement strategies eventually fail in most patients and are more ..

The primary pathophysiological change giving rise to the symptoms of Parkinson's disease (PD) is a loss of the dopaminergic neurons in the substantia nigra pars compacta (SNc) that are involved in modulating the function of basal ganglia (BG) nuclei. Unfortunately, traditional therapies for treatment of PD based on dopamine replacement strategies eventually fail in most patients and are associated with numerous side effects. A great deal of effort has been focused on developing a detailed understanding of the circuitry and function of the BG to develop novel, nondopaminergic, approaches for restoring normal BG function in PD patients. Exciting advances suggest that metabotropic glutamate receptors (mGluRs), including the group III mGluRs (mGluR4, -7 and -8), play important roles in regulating transmission through the BG and could serve as targets for novel PD therapeutics (Conn et al., 2005). For instance, mGluR4 activation reduces overactive GABA release at a specific inhibitory BG synapse (Macinnes and Duty, 2008; Marino et al., 2003; Valenti et al., 2003) and reverses motor deficits in a variety of rodent PD models (Konieczny et al., 2007; MacInnes et al., 2004; Marino et al., 2003; Ossowska et al., 2007; Valenti et al., 2003).

Basic protocol:1. Cells are plated at a density of 20,000 cells/well 1 day prior to assay in Thallium Assay Media (DMEM + 20mM HEPES, 10% dialyzed FBS, 1mM sodium pyruvate).2. The following day, media is removed from the cells.3. 20 ul/well of Fluo Zn dye (0.33 uM) in Assay Buffer (HBSS + 20mM HEPES) is added.4. The plates are incubated for one hour at RT.5. Dye is removed from the cells and the cell are washed with Assay Buffer leaving 20uL residual volume.6. Plates are incubated for 10 minutes at RT.7. Plates are loaded into a Hamamatsu FDSS.8. Compounds are tested for selectivity at a 10 uM final concentration (prepared as a 2X stock, 20 ul added per well) and the agonist is added at a 5X concentration (10 ul of agonist added per well, 50 uL total volume). A baseline read is taken for 1.5 seconds, the first addition (compound or DMSO-matched vehicle) occurs at 1.5 seconds and the second addition (agonist) is added at 142 seconds. The read continues for 300 seconds.Data analysis:Data were analyzed using Microsoft Excel. Raw data were opened in Excel and each data point in a given trace was divided by the first data point from that trace (static ratio). For experiments in which antagonists/potentiators were added, data were again normalized by dividing each point by the fluorescence value immediately before the agonist addition to correct for any subtle differences in the baseline traces after the compound incubation period. The slope of the fluorescence increase beginning five seconds after thallium/agonist addition and ending fifteen seconds after thallium/agonist addition was calculated. Curves were fitted using a four point logistical equation using Microsoft XLfit (IDBS, Bridgewater, NJ).This compound was a weak positive allosteric modulator of human mGluR6 and was assigned an 'Outcome' of 'Active' and a 'Score' of '50'.